The present application is the national stage under 35 U.S.C. 371 of international application PCT/IL99/00704, filed Dec. 28, 1999 which designated the United States, and which international application was published under PCT Article 21 (2) in the English language.
The present invention relates to antiviral compounds, more particularly to peptidomimetic conjugates of saccharides, such as aminoglycoside antibiotics, with acetamidino and guanidino compounds, and to antiviral, including antiretroviral, pharmaceutical compositions comprising them.
HIV: human immunodeficiency virus; RT: reverse transcriptase; RNAse: ribonuclease; UC781: a non-nucleoside RT inhibitor; AZT: azidothymidine; KS: Kaposi sarcoma; AIDS: acquired immunodeficiency syndrome; Tat: trans-activator of transcription; TAR: trans-activation responsive RNA region; LTR: long terminal repeat; P-TEFb: positive transcription elongation factor b; CDK9: cyclin-dependent kinase; ALX40-4C: D-arginine nonapeptide; CGP64222: peptide peptoid mimetic of Tat basic domain; HeLa: human epithelial cell line derived from cervical cancer; CXCR4: CXC (xcex1-chemotactic cytokines related to interleukin-8, containing C-X-C motif in their sequence, e.g. SDF-1xcex1) chemokine receptor 4; CD4: cluster of differentiation 4 (characteristic receptor of T-helper cells); CCR5: CC (xcex2-chemotactic cytokines, containing CC motif in their sequence) chemokine receptor 5; PBMC: peripheral blood mononuclear cells; T22: octadeca peptide, CXCR4 antagonist; AAC: aminoglycoside-arginine conjugates; R52: Tat-derived model undeca peptide, containing a single arginine moiety at position 52 of native Tat protein, in the strand of lysines; R4K: tetra-argininamido kanamycin A conjugate; R3G: tri-argininamido gentamicin C conjugate; MMP: xcex1-methyl D-mannopyranoside RMMP: mono-argininamido MMP conjugate; R4GC1a: tetra-argininamido gentamicin C1a isomer conjugate; GABA: xcex3-aminobutyric acid; GB4K: tetra-xcex3-(N-guanidino) butyramido-kanamycin A conjugate; NeoR: hexa-argininamido neomycin B conjugate; EIAV: equine infectious anemia virus; ED: equine dermal fibroblasts; DMF: dimethyl formamide; DCC: dicyclohexyl carbodiimide; M.p.: melting point; Pd/C: palladium on charcoal catalyst; TFA: trifluoro acetic acid; FABHRMS: fast atom bombardment high resolution mass spectroscopy; HSQC: heteronuclear single-quantum coherence; TOCSY: total correlation spectroscopy; RRE: Rev responsive RNA element; CAT: chloramphenicol acetyl transferase; DTT: dithiotreitol; EDTA: ethylenediamine tetraacetic acid; CI50: concentration of compound, that causes 50% inhibition of Tat-TAR interaction; CE50: concentration of 50% elution from affinity column; CD50: 50% binding concentration, related to Kd; Kd: dissociation constant; LAN-1: human neurioblastoma cell line; MPC-11: murine plasmocytoma cell line; MT-2, MT-4: human T-lymphoma cell lines, transfected with HTLV-I; HTLV-I, HTLV-II: Human-T-lymphoma virus type I or II; DMEM: Dulbecco modified Eagle""s medium; FCS: fetal calf serum; polybrene: hexadimetrine bromide; pfu: plaque forming unit; ELISA: enzyme-linked immuno sorption assay; P4-CCR5 MAGI: human cell line of monocyte/macrophages origin; HUVEC: human umbilical vascular endothelial cells; SUP-T1: human T-cell line; cpe: cytopathic effect; IC50: 50% inhibitory concentration; CC50: 50% cytotoxic concentration; EC50: 50% effective concentration; TI50: 50% in vitro therapeutic index (ratio CC50/EC50); SDS: sodium dodecyl sulfate; PAGE: polyacrylamide gel-electrophoresis; TLC: thin layer chromatography; HRP: horseradish peroxidase; SDF-1xcex1: stromal cell derived factor 1, subtype xcex1, the natural ligand of CXCR4; IL2: interleukin 2; IgG: immunoglobulin G; mAb: monoclonal antibody; 12G5: anti-CXCR4 mAb; 2D7: anti-CCR5 mAb; Leu3a: anti-CD4 mAb; PE: phycoerythrin; FITC: fluorescein isothiocyanate; RANTES: regulated on activation normal T-cell expressed and secreted chemokine; MPD: methyl pentandiol; SIR: single isomorphus replacement; SIRA: single anomalous replacement; MAD: multiple anomalous diffraction.
The transactivation responsive RNA (TAR) region of human immunodeficiency virus (HIV) long terminal repeat (LTR) regulates the viral gene expression via interaction with the HIV transactivator protein, Tat, and thus is an attractive target for drug design strategies (Gait and Karn, 1995). TAR is found at the 5xe2x80x2 end of all HIV-1 transcripts. It adopts a hairpin secondary structure consisting of a highly conservative hexanucleotide loop and a three-nucleotide bulge flanked by two double-stranded stems (Calnan et al., 1991 a, b). TAR is a positive enhancer that stimulates the synthesis of productive transcripts. It is unique in terms of eukariotic transcription control because it only functions as an RNA element. The activation by Tat is entirely dependent on the presence of the TAR RNA sequence. Tat activates expression by specific binding to TAR, which increases viral mRNA production several hundred-fold by stimulation of the elongation capacity of RNA polymerase II (Kingsman and Kingsman, 1996). HIV Tat binds the cyclin T subunit of P-TEFb and recruits P-TEFb to the HIV-1 LTR promoter. This process requires binding of Tat to the TAR bulge and of cyclin T to the TAR loop. The cyclin T associated CDK9 kinase then induces phosphorylation of the C-terminal domain of RNA polymerase II, and of other polymerase II-associated proteins, leading to the transition from non-processive to processive transcription (Cullen, 1998).
Binding of Tat protein to TAR is mediated by the nine amino acid region RKKRRQRRR (residues 49-57) of the protein (e.g. Calnan et al., 1991 a, b; Churcher et al., 1993). The nona-arginine peptide (R9) binds to TAR with the same affinity and specificity as the above wild-type Tat peptide, whereas the nona-lysine peptide (K9) binds to TAR non-specifically and with a ten-fold lower affinity. The R9-containing Tat mutant protein gives wild-type trans-activation activity and is 100-fold more active than the K9-containing protein. Insertion of a single arginine moiety at position 52 or 53 (synthetic peptides R52 of the sequence YKKKRKKKKA or R53) restores RNA-binding affinity and specificity of the peptide as well as its trans-activation potency (Calnan et al., 1991 b). Mutagenesis studies on TAR RNA demonstrated that the bulge (U23-C24-U25) and two base pairs at both sides of the bulge (e.g. Cordingley et al,. 1990; Roy et al, 1990; Delling et al., 1992) are important for Tat binding. Full length Tat protein binds TAR with only moderate affinity and specificity in vitro. The first 37 N-terminal amino acids of the Tat protein decrease its affinity to TAR in comparison to the specific recognition Tat (38-72) peptide (Rana and Jeang, 1999). It was shown that human cyclin T1 promotes cooperative binding of Tat protein to TAR RNA in vitro and mediates trans-activation in vivo. Although cyclin T1 does not bind TAR RNA, it may interact with the TAR loop in a ternary complex of cyclin T1-Tat-TAR (Wei et al., 1998).
NMR structures of HIV TAR bound to different ligands, e.g. as peptides that mimic the basic region of Tat, arginine and arginineamide, show that the ligands bind to the TAR RNA major groove (Puglisi et al., 1992, 1993; Aboul-ela et al., 1995, 1996). The bulge structure allows ligands to access the major groove of TAR, which induces folding in the bulge and formation of unusual base-triples (Puglisi et al., 1992, 1993, Aboul-ela et al., 1995, 1996). The TAR RNA hairpin can adopt two major conformations. In the absence of ligands, the bulge nucleotides stack within the RNA stem, severely distorting its helical continuity (Puglisi et al., 1992; Aboul-ela et al., 1996). When either L-arginineamide or the Tat peptide bind to TAR, the bulge nucleotides loop out of the remaining stem, allowing the upper and the lower stem helices to stack coaxially (Puglisi el al., 1992, 1993). The NMR structure of L-arginineamide bound to TAR suggests proximity between the bulge and apical loop across the RNA major groove (Aboul-ela et al., 1995, 1996). The specific interactions between HIV Tat protein and TAR RNA are still unknown but could be modeled as a basic ax-helix of Tat peptide lying in the major groove of TAR (Mujeeb et al., 1996).
Tat-derived basic peptides, as well as the oligocationic peptide and peptoid Tat mimetics bind TAR RNA with high affinity in vitro (e.g . Calnan et al., 1991 a, b; O""Brien et al., 1996; Hamy et al., 1997; Huq et al. 1997, 1999 a). Tat-mimetic compounds ALX40-4C (O""Brien et al., 1996) and CGP64222 (Hamy et al., 1997), that target TAR RNA, demonstrate a pronounced antiviral activity. D-Tat peptide, derived from Tat 37-72 sequence, binds to TAR RNA major groove and interferes with the transcriptional activation by Tat protein in vitro and in HeLa cells (Huq et al., 1999 a).
The functions of Tat protein in viral progeny are not limited to HIV trans-activation event. Tat trans-activates a number of cellular genes and acts as chemokine analogue, while secreted extracellularly (Albini et al., 1998). Tat induces positive chemotaxis of human monocytes and monocyte-derived dendritic cells (Benelli et al, 1998). Extracellular Tat protein was shown to up-regulate the expression of CXCR4 chemokine receptor in primary-resting CD4+ T-cells (Secchiero et al., 1999) as well as CCR5 receptor on monocytes (Weiss et al., 1999), which serve as co-receptors of viral entry for T-tropic and M-tropic HIV strains, respectively (Berger et al., 1999). It was shown, that HIV Tat protein, released by infected cells, differentially induces CXCR4 and CCR5 expression in peripheral blood mononuclear cells (PBMC), and promotes infectivity of both M- and T-tropic HIV-1 strains (Huang et al., 1999). The discovery of chemokine receptors as cofactors involved in the entry of HIV in the host cell has renewed the interest in the early steps of virus replication as a target for therapeutic intervention. A number of compounds have been described to interact with CCR5, the chemokine receptor used by macrophage-tropic (MT, R5) strains of HIV (Simmons et al., 1997). Two other groups have also described newly identified CXCR4 antagonists: ALX40-4C (Doranz et al., 1997) and T22 (Murakami et al., 1997), an octadecapeptide with 8 positive charges or its derivatives (Arakaki et al., 1999).
Another function of Tat protein (in synergism with inflammatory cytokines) is in induction of angiogenesis and the development of Kaposi sarcoma in AIDS patients (Barillari et al., 1999). All the above Tat functions are dependent on the presence of the basic domain in the protein structure. However, Tat has multiple domains, and two of them mediate the cellular and viral effects of extracellular Tat. Peptides, derived from cystein-rich domain and (possibly in combination with) basic domain were found to mimic the effects of a whole Tat protein in HIV-infected cells (Boykins et al., 1999).
Among natural RNA targeting molecules, aminoglycoside antibiotics have interesting properties that make them similar to peptide RNA binders. They are known to bind efficiently to RNA, such as 16S RNA or intrones type I (von Ahsen et al. 1992). Neomycin B and tobramycin inhibit HIV Rev-RRE interaction in vitro at concentrations of 1-10 xcexcM, whereas kanamycin and gentamicin do not display any inhibition at 100 xcexcM. Recent experiments have demonstrated that among the aminoglycoside antibiotics, neomycin has the greatest inhibitory effect on Tat binding to TAR in vitro in the range of 1-100 xcexcM. This phenomenon was attributed to the direct association of the aminoglycoside antibiotic with TAR RNA in its lower stem (Wang et al., 1998).
It has now been found, in accordance with the present invention, that by combining a carbohydrate skeleton, either a mono or an oligosaccharide similar to aminoglycoside antibiotics with side-chains of variable length bearing a guanidine moiety or a chemical group with a similar geometry and/or charge properties resembling peptide side chains, a new class of peptidomimetic TAR RNA binders is obtained that are anti-HIV compounds and suppress viral replication (HIV-1 and EIAV) by inhibiting transactivation by Tat as well as by blocking viral entry to cells through chemokine-receptor-dependent mechanism. These relatively low molecular weight compounds, which mimic the functions of Tat protein basic domain, are the first examples of substances composed of a carbohydrate core conjugated to L-arginine or similar compounds with side chains, bearing guanidino or acetamidino groups that efficiently bind to TAR RNA as well as efficiently inhibit HIV-1 entry to T-cells.
The present invention thus relates to conjugates of saccharides and acetamidino or guanidino compounds of the formula I: 
xe2x80x83wherein
A is CH3 or NH2; X is a linear or branched C1-C8 alkyl chain optionally containing hydroxy, amino and/or oxo groups; n is an integer from 1 to 6, and Sac is the residue of a mono- or oligo-saccharide.
When A is CH3, the conjugates are acetamidino saccharide conjugates, and when A is NH2, the conjugates are guanidino saccharide conjugates. Sac may be the residue of a monosaccharide, in which case n is 1-5, or of an oligosaccharide, in which case n is 1-6.
Examples of monosaccharide conjugates according to the invention are methyl 6-deoxy-6-(N-acetamidino)-xcex1-D-mannopyranoside, methyl 6-deoxy-6-guanidino-xcex1-D-mannopyranoside and methyl 6-deoxy-6-(N-L-argininamido)-xcex1-D-mannopyranoside (the compounds 11, 10 and 12 herein) of the formulas shown in Scheme 1 herein.
The natural or synthetic oligosaccharides are, for example, an aminoglycoside antibiotic such as, but not limited to, neomycin, kanamycin or gentamicin, or synthetic oligosaccharides. The aminoglycoside-arginine conjugates of the invention will also sometimes be designated herein in the specification as AAC.
The invention further provides pharmaceutical compositions comprising a conjugate of the invention and a pharmaceutically acceptable carrier, particularly for use as antiviral, more particularly as antiretroviral, compositions, alone or together with other anti-AIDS agents such as AZT or protease inhibitors.